Metabolic adaptation to oxidative stress is mediated through P5CS oxidation

Cancer cells are known to produce high amount of reactive oxygen species (ROS). ROS are increasingly recognized as important signaling molecules through oxidation of protein cysteine residues.

SICyLIA, a simple, unbiased, and quantitative proteomic method which detects proteome-wide cysteine oxidation, was used to analyze both chronic and acute oxidative stress. Immortalized primary mouse kidney cells deficient in Fumarate Hydratase (FH), a tricarboxylic acid (TCA) cycle enzyme, that display elevated levels of ROS, were used as a model for chronic oxidative stress. Control wild-type cells treated with hydrogen peroxide were used for acute oxidative stress. SICyLIA identified cysteine oxidation in Pyrroline-5-Carboxilate Synthase (P5CS), an enzyme required for proline synthesis, under both stress conditions. Interestingly, P5CS enzymatic reaction shares an intermediate - glutamyl-5-phoshate - with glutathione synthesis pathway potentially linking P5CS modification to oxidative stress regulation. Indeed, metabolomic analysis with ¹³C-labeled glutamine demonstrated a decrease in proline synthesis and an increase in 5-oxoproline (a stable cyclic form of glutamyl-5-phoshate) and in glutathione synthesis in both hydrogen peroxide-treated cells and FH-deficient cells. These changes indicate a switch between proline synthesis and glutathione synthesis pathways under oxidative stress. Concomitantly, P5CS-deficient cells displayed decreased resistance to hydrogen peroxide treatment compared with wild-type cells, even when exogenous proline was supplied. Moreover, FH-deficient cells displayed increased resistance to ROS-generating treatments, including ionizing radiation.

We suggest that the channeling of glutamyl-5-phoshate from proline to glutathione biosynthesis after P5CS oxidation may play an important role in protecting cells from oxidative stress.